This invention relates to the use of highly water soluble, uncharged, safe materials to create reversible intracellular hypertonicity as a means of incorporating desired materials into red blood cells or achieving therapeutically desirable changes in the characteristics of intracellular hemoglobin. A particular utility relates to its ability to reduce the mean corpuscular hemoglobin concentration, thereby inhibiting the precipitation or gelation of abnormal hemoglobins such as S hemoglobin and the destruction of cells by sickling.
In a specific embodiment the method of the present invention effects the introduction of materials into red blood cells by subjecting red blood cells which have been separated from whole blood to incubation with dimethyl sulfoxide or glycerol, and to a transient osmotic shock in the presence of an organic phosphate (which decreases the affinity of hemoglobin for oxygen) in a diluent solution as a result of which the cell membranes are rendered more permeable or porous, thus permitting passage of the organic phosphate into the cells. The osmotic balance is then restored and the cells resume their normal shape.
As is well known, hemoglobin (hereinafter Hb) in red blood cells (hereinafter RBC) transports oxygen from the lungs to body tissues. The normal oxygen-Hb dissociation curve (representing the reversible oxy- and deoxy-Hb equilibrium) indicates that Hb is substantially completely saturated at the oxygen partial pressure of the alveoli and substanially less than saturated at normal oxygen partial pressures in tissues which are adequate to allow aerobic metabolism.
Conditions can occur which cause a "left shift" in the oxygen-Hb dissociation curve, i.e., the Hb develops a stronger affinity for oxygen, resulting in decreased oxygen release at the normal oxygen partial pressure of tissue. For example, blood stored under blood bank conditions undergoes a left shift due to disappearance of naturally occurring 2,3 Bis phosphoglycerate (2,3 DPG), which binds reversibly to Hb and reduces its oxygen affinity.
Other compounds are known to have an even stronger effect, viz., phosphorylated inositols. Inositol hexaphosphate (hereinafter IHP) is reported to have the strongest "right shift" effect on the Hb dissociation curve. IHP is a commonly occurring plant product also known as phytic acid. However, IHP cannot penetrate the membranes of RBC, and if administered in vivo, will precipitate because of its strong affinity for calcium ions. Accordingly, incorporation of IHP intracellularly in RBC can be effected only by special techniques.
An article by G. I. Dale et al, Biochem Med 18:220-225 (1977 ) describes a previously known method for incorporating materials into RBC which involves rupturing RBC by osmotic swelling. This is done by suspending RBC in a hypotonic solution. Thereafter the osmotic pressure is returned to isotonic which allows the RBC membranes to reseal. All such methods suffer from the disadvantage of loss of substantial portions of the cell contents, particularly Hb, which decreases the integrity and viability of the cells reformed by resealing of membranes treated by these methods.
U.S. Pat. No. 4,192,869, issued to Nicolau and Gersonde , discloses the interaction of RBC with liposomes containing an allosteric effector, particularly IHP.
European patent application published Nov. 24, 1982 as EP No. 0065 292, in the names of Gersonde and Weiner, describes a method of freeze-preserving preparations of lipid vesicles containing allosteric effectors for eventual thawing and use.
The method of U.S. Pat. No. 4,192,869 requires the use of lipid materials (derived from sources outside the organism being treated), which may be incorporated into RBC membranes. In actual practice it has been found that the method of this patent produces inconsistent results and unpredictable failures to incorporate IHP into RBC. A number of variables, such as degree of aeration, pH control, and timing and volume of buffer washes, have been found to affect the degree of IHP incorporation, but the interdependence and possible criticality of these parameters has not been determined.
Other disclosures relating to introduction of allosteric effectors into RBC include:
M. Weiner, "Right Shifting of Hb-O.sub.2 Dissociation in Viable Red Cells by Liposomal Technique" Biol. Cell, 47, 65-70 (1983); and
R. S. Franco, M. Weiner, K. Wagner and O. J. Martelo, "Incorporation of Inositol Hexaphosphate into Red Blood Cells Mediated by Dimethyl Sulfoxide" Life Sciences, 32, 2763-2768 (1983).
Numerous articles are referenced in the above publications which are of background interest.